The effects of a novel membrane-penetrable modulator, 2APB (2-aminoethoxy diphenyl borate), on Ins(1,4,5)P₃-induced Ca²⁺ release were examined. 2APB inhibited Ins(1,4,5)-P₃-induced Ca²⁺ release from rat cerebellar microsomal preparations without affecting [³H]Ins(1,4,5)P₃ binding to its receptor. The IC₅₀ value (concentration producing 50% inhibition) of 2APB for inhibition of Ins(1,4,5)P₃ (100 nM) induced Ca²⁺ release was 42 μM. Further increase in the concentration of 2APB (more than 90 μM) caused a gradual release of Ca²⁺ from cerebellar microsomal preparations. Addition of 2APB to the extracellular environment inhibited the cytosolic Ca²⁺ ([Ca²⁺]_c) rise in intact cells such as human platelets and neutrophils stimulated by thromboxane-mimetic STA₂, or thrombin, and leukotriene B₄ (LTB₄) or formyl-methionine-leucine-phenylalanine (FMLP), respectively. 2APB inhibited the contraction of thoracic aorta isolated from rabbits induced by angioten-sin II (AII), STA₂, and norepinephrine in a non-competitive manner, but showed no effect on the contraction of potassium-depolarized muscle. 2APB had no effect on the Ca²⁺ release from the ryanodine-sensitive Ca²⁺ store prepared from rat leg skeletal muscle and heart. Although the specificity of 2APB with respect to the intracellular signaling system was not fully established, 2APB is the first candidate for a membrane-penetrable modulator of Ins(1,4,5)P₃ receptor, and it should be a useful tool to investigate the physiological role of the Ins(1,4,5)P₃ receptor in various cells.